This invention relates to an improved synthesis of derivatives of N-acetylneuraminic acid monoalkylated at either the 4- or the 7-position. The synthetic procedures represent improvements and enhancements that permit obtaining large quantities of the products suitable for commercial production. These derivatives of N-acetylneuraminic acid can be used as chromogenic substrates for the detection of viral neuraminidases.
Viral infections are a principal cause of illness due to communicable diseases that affect the public at large. Of these, influenza viruses, including types A and B, are a significant factor responsible for causing respiratory symptoms as well as systemic malaise; other respiratory viruses include parainfluenza 1, 2, 3, and 4, respiratory syncytial virus, and adenovirus. The influenza viruses undergo rapid mutation of strains, producing pathogens with varying degrees of virulence and severity of symptoms. Recently, influenza infection has been as high as the fifth leading cause of death from acute respiratory disease in the United States (Morbidity and Mortality Weekly Report, 36 (1987) 2).
Influenza virus types A, B, and C belong to the family of Orthomyxoviridae. Influenza A and B are significant pathogens in children and adults causing sever lower respiratory tract disease, whereas influenza C can cause sporadic upper respiratory tract. illness. Influenza virus is highly contagious and can affect large proportions of the population each winter. Influenza A epidemics occur every 2-3 years, whereas influenza B epidemics appear every 4-6 years. Symptoms include moderate to high fever together with chills, headache, myalgia, rhinorrhea, among others. Importantly, virus progeny are detectable 24 hours prior to the appearance of symptoms, and virus titers peak 24-48 hours after symptoms arise.
For this reason it is important to have available ways of diagnosing the presence of an influenza infection, and of distinguishing it from related viral and bacterial infections. Particularly among infants, the elderly and those having compromised or deficient immune responses, early diagnosis of influenza can lead to appropriate symptomatic treatment to minimize the risk of morbidity.
Diagnosis of viral infection, such as infection by influenza virus, may be carried out by detecting the presence of unique moieties characteristic of the virus. Virus particles typically carry distinctive antigenic components on the exterior of the virion which may be detected by specific ligand-antiligand interactions, in particular by the use of an antibody specific for a viral epitope. Such interactions rely on the law of mass action, and for this reason may have limited sensitivity. Many virus particles additionally carry specific enzymatic activities on the virion particle. Influenza viruses, parainfluenza viruses, and mumps are examples of such viruses; they are endowed with a virus-specific surface glycoprotein with neuraminidase activity as an integral part of the virion. Utilization of the enzymatic activity for diagnostic assays in such cases offers the potential for increasing the sensitivity of a detection method. For example, influenza A and B, having neuraminidase activity, are detectable in this way, whereas influenza C is not.
N-acetylneuraminic acid (sialic acid, Neu5Ac), whose structure is shown below with atoms numbered, is the terminal saccharide residue 
of many complex carbohydrate side chains of cell surface glycoproteins. In this structure the C2 position is the anomeric carbon, which is characterized by being part of a hemiketal group, or the 2-ketoside moiety. The glycosidic linkage bonding Neu5Ac with the penultimate saccharide is the substrate of the neuraminidase activity of the influenza virion. The neuraminidase hydrolyzes glycosidic linkages having the a anomeric configuration, thereby cleaving Neu5Ac from the penultimate saccharide. Consequently, suitable synthetic substrates may be derivatives of Neu5Ac in 2-ketosidic a linkage with a detectable moiety. The moiety then provides a product, when the substrate is acted upon by the viral neuraminidase activity, that signals the presence and amount of influenza virus particles in a sample. Since the viral enzyme cleaves the substrate catalytically, the sensitivity of detecting the presence of the enzyme is greatly enhanced. For this reason the overall sensitivity of detection may be comparable to, and may even be improved over, that provided by antibody binding assays.
One method for detecting the presence of a virus through the reaction of an enzyme with a chromogenic substrate for the enzyme is described in U.S. Pat. No. 5,252,458, which is incorporated herein by reference. An assay for the direct measurement of influenza neuraminidase was developed by Yolken et al. (J. Infectious Diseases 142 (1980) 516-523). Yolken et al. used the 4-methylumbelliferyl-2-ketoside of Neu5Ac as a fluorescent substrate to measure neuraminidase activity in preparations containing small quantities of cultivated virus as well as in some nasal wash specimens from human volunteers infected with the influenza virus. Yolken et al. suggested that xe2x80x9csuccessful development of influenza neuraminidase might thus provide for a practical means of influenza diagnosis that is sufficiently rapid to allow for the institution of appropriate preventive and therapeutic interventions.xe2x80x9d According to Yolken et al., colorimetric assays were insufficiently sensitive for clinical applications, suggesting instead that fluorimetric assays for influenza neuraminidase might be suitable for detecting the virus in clinical samples.
Pachucki et al. (J. Clinical Microbiology 26 (1988) 2664-2666) tested the 4-methylumbelliferyl-2-ketoside of Neu5Ac on clinical specimens collected from influenza patients. Due to its low sensitivity, the assay was not useful in detecting neuraminidase in clinical specimens. The assay did, however, identify 91% of virus-positive isolates 25 hours after inoculation of tissue cultures.
The use of modified Neu5Ac substrates can increase the specificity of the neuraminidase assay. In sialic acids, the C4 position has been reported to play an important role in enzyme-substrate interactions. Further, since it is known that salivary bacterial enzymes exhibit neuraminidase activity (Varki et al., J. Biol. Chem. 258 (1983) 12465-12471), it is essential to avoid these undesired enzymatic activities. It has, for example, been shown that ketosides of 4-methoxy-Neu5Ac are resistant towards certain bacterial sialidases (Beau et al., Eur. J. Biochem. 106 (1980) 531-540).
U.S. Pat. No. 5,252,458 to Liav et al. provides a direct chromogenic assay for detecting a virus, including influenza viruses and parainfluenza viruses, that include in the virion a characteristic enzymatic activity, such as neuraminidase activity. The method, which is implemented in a clinic or physician""s office, includes incubating a clinical sample suspected of containing the virus with a solution of a chromogenic substrate. The samples typically are obtained by swabbing the pharyngeal, or nasopharyngeal surfaces. If the virus is present, a chromogen is cleaved from the substrate, the chromogen is then reacted with a precipitating agent that intensifies the color, and the colored precipitate is concentrated for detection as a colored spot. The patent also discloses a kit for use in the clinic or physician""s office that includes a filtration device for concentrating the colored precipitate into a spot.
U.S. Pat. No. 5,252,458 to Liav et al. provides synthetic routes for the synthesis of a precursor for chromogenic substrates that are useful in the diagnostic assay of viruses. Specifically the patent discloses syntheses for 4-alkoxy-N-acetylneuraminic acid.
U.S. Pat. No. 5,663,055 to Turner et al. discloses 4-modified Neu5Ac chromogenic substrates of viral neuraminidases for use in assays carried out in a clinic or a physician""s office. The modification at position 4 includes hydrogen, fluorine, methoxy or ethoxy, and the substrate is sensitive to neuraminidases from influenza and parainfluenza viruses. Clinical samples are used in the assays, and distinct colors are produced as a result. The samples are typically pharyngeal, nasopharyngeal or respiratory secretions collected from patients as wash, swab, or expectorate specimens. U.S. Pat. No. 5,663,055 describes syntheses of several 4-modified derivatives of Neu5Ac, including 4-methoxy, 4-deoxy, and 4-fluoro derivatives. It further discloses syntheses for chromogenic ketosides of such 4-modified derivatives of Neu5Ac. This patent further establishes that 4-O-methyl chromogenic ketosides of Neu5Ac are selective for influenza virus A and for influenza virus B neuraminidases. These ketosides are insignificantly reactive with bacterial neuraminidases from the oral cavity, and are not significantly hydrolyzed by mammalian neuraminidase.
U.S. Pat. No. 5,719,020 to Liav et al. discloses 4,7-di-O-alkylated chromogenic ketosides of N-acetylneuraminic acid for use as the substrate in assays that are specific for influenza A and influenza B. The assays are performed in the clinic or physician""s office. The chromophoric product may be precipitated and the colored precipitate concentrated for detection. Assays are also disclosed that permit discriminating between influenza A and influenza B on the one hand, and other viruses.
Although modification of the 4-position of Neu5Ac""s provides specificity between certain viral and certain bacterial neuraminidase activities, it is still desirable to obtain substrates which provide enhanced specificity between the various viral neuraminidase reactivities while maintaining the specificity between viral and bacterial neuraminidase activities. Such substrates allow, for example, high specificity for particular types of neuraminidase-containing viruses and allow better and more directed treatment regimes. Use of specific substrates thus allows for more accurate surveillance of viral infections and more focused medical intervention as appropriate. U.S. Pat. No. 5,719,020, incorporated herein by reference, provides chromogenic and fluorogenic 4,7-disubstituted N-acetylneuraminic acid substrates for viral neuraminidase activities. These derivatives provide further specificity or differentiation between the various viral neuraminidase activities while maintaining the specificity between viral and bacterial neuraminidase activities. In particular, in U.S. Pat. No. 5,719,020 it is shown that the 4,7-modified Neu5Ac chromogenic substrates disclosed therein distinguish between influenza type A and B viruses on the one hand, and neuraminidases from other viral and bacterial pathogens on the other. A chromogenic derivative of 4-O-methyl Neu5Ac, however, develops color when exposed to parainfluenza virus types 1, 2, and 3, and mumps, in addition to influenza types A and B. Certain reactions leading to the synthesis of 4,7-di-O-alkyl Neu5Ac chromogenic ketosides disclosed in U.S. Pat. No. 5,719,020 likewise are inefficient and provide poor purity and low yields.
The synthetic procedures leading to various 4-O-alkyl Neu5Ac derivatives are disclosed in U.S. Pat. No. 5,556,963, but have certain difficulties associated with them. These include the provision of synthetic routes involving a large number of distinct chemical reactions. These require an excessive number of unit operations, leading to the need for many reagents, solvents, pieces of laboratory apparatus, and for extended time to obtain the product. Additionally, with respect to the objectives of the present invention, the syntheses in U.S. Pat. No. 5,556,963 do not provide the 4-O-alkyl Neu5Ac chromogenic ketosides that serve as substrates for viral neuraminidases. The syntheses of 4-O-alkyl Neu5Ac chromogenic ketosides described in U. S. Pat. No. 5,663,055 are generally cumbersome and inefficient, involving, for example, the need to transfer the reactant between aqueous and organic phases using a phase transfer reagent.
Zbiral et al. (Liebigs Ann. Chem 1989:519-526) studied the 4-methylumbelliferyl xcex1 ketosides of 7-epi Neu5Ac, 7,8-(bis)epi Neu5Ac, 7-deoxy Neu5Ac and 4,7-dideoxy Neu5Ac, among others, as substrates for neuraminidase from Vibrio cholerae. It was shown that the glycosidic hydrolysis was significantly slowed only for 7,8-(bis)epi Neu5Ac, 7-deoxy Neu5Ac and 4,7-dideoxy Neu5Ac of those derivatives tested. Zbiral et al. (Monatshefte Chem. 119:127-141 (1988)) synthesized 7-, 8-, and 9-, monodeoxy derivatives of Neu5Ac and 4,7-dideoxy Neu5Ac with the objective of studying their activation by cytidine monophosphate-sialate synthase. U.S. Pat. No. 5,719,020 reports that 5-bromo-3-indolyl 4,7-di-O-methyl Neu5Ac ketoside is selectively hydrolyzed by the neuraminidase activity of only influenza types A and B, whereas 5-bromo-3-indolyl 4-O-methyl Neu5Ac ketoside is acted upon by influenza types A and B, as well as by the neuraminidase activity of parainfluenza types 1 and 2, and mumps, but not significantly by the enzyme of parainfluenza type 3, and not at all by respiratory syncytial adenovirus. Thus there is important selectivity manifested at the 4- and 7-positions of Neu5Ac which suggests that 7-O-alkyl Neu5Ac chromogenic ketosides afford the potential of considerable selectivity among bacterial and viral neuraminidases.
The present invention addresses the deficiencies identified above related to the preparation of 4-alkyl chromogenic glycosides and 7-alkyl chromogenic glycosides of N-acetylneuraminic acids. As set forth in detail herein, the number of synthetic steps and attendant number of unit operations is reduced. The use of chromatographic separations likewise is reduced to the extent possible. Additionally, the synthetic reactions have been optimized and the recovery procedures enhanced to provide high step yields and thereby high overall yields for the final products that are unexpected by the present state of knowledge in the field. The resulting improvements permit economical preparation of viral neuraminidase substrates on the scale of kilograms for commercial sale.
The present invention discloses a method of preparing a 4-O-alkyl chromogenic ketoside of N-acetylneuraminic acid (Neu5Ac)that includes the steps of:
(a) alkylating Neu5Ac on its C1 carboxyl and anomeric C2 hydroxyl to provide an alkyl ester alkyl ketoside derivative of Neu5Ac;
(b) protecting the 8- and 9-hydroxyl groups of the alkyl ester alkyl ketoside derivative of Neu5Ac to form a protected alkyl ester alkyl ketoside derivative of Neu5Ac;
(c) alkylating the 4-hydroxyl group of the protected alkyl ester alkyl ketoside derivative of Neu5Ac by contacting it with a composition containing an alkyl halide to form a 4-O-alkyl protected alkyl ester alkyl ketoside derivative of Neu5Ac;
(d) selectively deprotecting the 8- and 9-hydroxyl groups and dealkylating the 2-hydroxyl group of the ketoside derivative obtained in step (c), and derivatizing the resulting 2-, 7-, 8-, and 9-hydroxyl groups;
(e) activating the anomeric carbon of the product obtained in step (d) to form a 4-O-alkyl alkyl ester derivative of Neu5Ac, derivatized at the 7-, 8- and 9-hydroxyls, and activated at the anomeric carbon;
(f) contacting the product obtained in step (e) with a suspension of a chromogenic moiety in a solvent to form a chromogenic ketoside of a 4-O-alkyl alkyl ester derivative of Neu5Ac, derivatized at the 7-, 8- and 9-hydroxyls; and
(g) obtaining a 4-O-alkyl chromogenic ketoside of Neu5Ac from the derivatized chromogenic ketoside obtained in step (f).
In important embodiments of the method, protecting the 8- and 9-hydroxyl groups described in step (b) involves forming a ketal or an 8,9-epoxide. In additional important embodiments, the alkyl halide employed in step (c) is methyl iodide and the composition further comprises either silver oxide or a mixture of barium oxide and barium hydroxide.
In significant embodiments of the method, the activation of the anomeric carbon described in step (e) involves placing a halo substituent, such as chloro, on the anomeric carbon.
In further advantageous embodiments, the chromogenic moiety described in step (f) is chosen from a wide variety of chromogenic, luminescent, or chemiluminescent moieties, and in a more advantageous embodiment, the chromogenic moiety is 5-bromo-3-indolyl.
In additional significant embodiments of the method, alkyl groups on the C1 carboxyl, the anomeric hydroxyl, and the 4-position are the same or different and are chosen from the group consisting of methyl, ethyl, propyl, isopropyl, and normal or branched butyl; more significantly, they are all methyl groups.
In a preferred embodiment, a method of preparing a 4-O-methyl chromogenic ketoside of N-acetylneuraminic acid (Neu5Ac) is presented, including the following sequential steps:
(a) contacting Neu5Ac with methanol and the protonated form of a cation exchange resin to methylate the C1 carboxyl group and the C2 hydroxyl group, forming a methyl ester methyl ketoside of Neu5Ac;
(b) contacting the methylated ester ketoside of Neu5Ac provided in step (a) with acetone and p-toluenesulfonic acid, to form a 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac;
(c) contacting the 8,9-isopropylidene methylated ester ketoside of Neu5Ac provided in step (b) with methyl iodide and silver oxide to form a 4-O-methyl 8,9-isopropylidene methylated ester ketoside of Neu5Ac;
(d) selectively removing the 8,9-isopropylidene and C2 methyl groups from the ketoside product obtained in step (c) and acetylating the anomeric, 7-, 8- and 9-hydroxyl groups of the resulting intermediate;
(e) activating the anomeric carbon of the product obtained in step (d) by converting the acetate to chloride;
(f) contacting the activated chloride product provided in step (e) with a suspension of a derivative of 5-bromo-3-indolol and potassium tert-butoxide in a solvent to form a chromogenic ketoside of a 4-O-methyl methyl ester of Neu5Ac, acetylated at the 7-, 8- and 9-hydroxyls; and
(g) deblocking the acetyl groups at the 8- and 9-hydroxyl groups and the methyl ester from the chromogenic ketoside of Neu5Ac obtained in step (f), to form the 4-O-methyl chromogenic ketoside of Neu5Ac.
In another preferred embodiment, a method of preparing a 4-O-methyl chromogenic ketoside of N-acetylneuraminic acid (Neu5Ac) includes the following sequential steps:
(a) contacting Neu5Ac with methanol and the protonated form of a 5cation exchange resin to methylate the C1 carboxyl group and the C2 hydroxyl group, forming a methyl ester methyl ketoside of Neu5Ac;
(b) contacting the methylated ester ketoside of Neu5Ac provided in step (a) with
(i) an acid chloride of an organic sulfonic acid, and
(ii) treating the result obtained in step (b) (i) with a composition containing sodium methoxide and methanol, providing an 8,9-epoxide methyl ester methyl ketoside of Neu5Ac;
(c) methylating the 4-hydroxyl group of the 8,9-epoxide methylated ester ketoside of Neu5Ac provided in step (b) by a process chosen from the group consisting of
(i) contacting the epoxide ketoside with a composition containing methyl iodide and silver oxide,
(ii) contacting the epoxide ketoside with a composition containing dimethylsulfate and sodium hydride, and
(iii) a sequential combination of step (c)(i) and step (c)(ii), providing a 4-O-methyl 8,9-epoxide methylated ester ketoside of Neu5Ac;
(d) selectively removing the 8,9-epoxide and C2 methyl groups of the ketoside product obtained in step (c) and acetylating the anomeric, 7-, 8- and 9-hydroxyl groups of the resulting intermediate;
(e) activating the anomeric carbon of the product obtained in step (d) by converting the acetate to chloride;
(f) contacting the activated chloride product provided in step (e) with a suspension of a derivative of 5-bromo-3-indolol and potassium tert-butoxide in a solvent to form a chromogenic ketoside of a 4-O-methyl methyl ester of Neu5Ac, acetylated at the 7-, 8- and 9-hydroxyls; and
(g) deblocking the acetyl groups at the 8- and 9-hydroxyl groups and the methyl ester from the chromogenic ketoside of Neu5Ac obtained in step (f), to form the 4-O-methyl chromogenic ketoside of Neu5Ac.
The present invention further provides a method of preparing a 7-O-alkyl chromogenic ketoside of N-acetylneuraminic acid (Neu5Ac) that includes the following steps in order:
(a) alkylating Neu5Ac on the C1 carboxyl and C2 (anomeric) hydroxyl to provide an alkyl ester alkyl ketoside derivative of Neu5Ac;
(b) protecting the 8- and 9-hydroxyl groups of the alkyl ester alkyl ketoside derivative of Neu5Ac to form a protected alkyl ester alkyl ketoside derivative of Neu5Ac;
(c) blocking the 4-hydroxyl group of the protected alkyl ester alkyl ketoside derivative of Neu5Ac with a blocking group to form a blocked protected alkyl ester alkyl ketoside derivative of Neu5Ac;
(d) alkylating the 7-hydroxyl group of the blocked protected alkyl ester alkyl ketoside derivative of Neu5Ac by contacting the derivative with a composition comprising an alkyl halide to form a 7-O-alkyl blocked protected alkyl ester alkyl ketoside derivative of Neu5Ac;
(e) deblocking the 7-O-alkyl blocked protected alkyl ester alkyl ketoside derivative of Neu5Ac to form a 7-O-alkyl protected alkyl ester alkyl ketoside derivative of Neu5Ac;
(f) selectively deprotecting the 8- and 9-hydroxyl groups and dealkylating the 2-hydroxyl group of the ketoside product obtained in step (e), and derivatizing the 2-, 4-, 8-, and 9-hydroxyl groups of the resulting product;
(g) activating the anomeric carbon of the product obtained in step (f) to form a 7-O-alkyl alkyl ester derivative of Neu5Ac, derivatized at the 4-, 8- and 9-hydroxyls, and activated at the anomeric carbon;
(h) contacting the product obtained in step (g) with a suspension of a chromogenic moiety in a solvent to form a chromogenic ketoside of a 7-O-alkyl alkyl ester derivative of Neu5Ac, derivatized at the 4-, 8- and 9-hydroxyls; and
(i) obtaining a 7-O-alkyl chromogenic ketoside of Neu5Ac from the derivatized chromogenic ketoside obtained in step (h).
In advantageous embodiments of the method, protecting the 8- and 9-hydroxyl groups described in step (b) includes forming a ketal or an 8,9-epoxide. Additionally, in an important embodiment, the blocking group in step (c) is benzyl.
In a significant embodiment, the activation of the anomeric carbon described in step (g) includes placing a halo substituent, preferably a chloro, on the anomeric carbon. In further significant embodiments, a wide variety of chromogenic moieties may be employed in step (h); preferably the chromogenic moiety is 5-bromo-3-indolyl.
In further important embodiments, the alkyl group on the C1 carboxyl, the alkyl group on the anomeric hydroxyl, and the alkyl group at the 7-position are the same or different and are chosen from among methyl, ethyl, propyl, isopropyl, and normal or branched butyl; preferably all three groups are methyl.
The invention further provides a method of preparing a 7-O-methyl chromogenic ketoside of N-acetylneuraminic acid (Neu5Ac) that includes the following sequential steps:
(a) contacting Neu5Ac with methanol and the protonated form of a cation exchange resin to methylate the C1 carboxyl group and the C2 hydroxyl group, forming a methyl ester methyl ketoside of Neu5Ac;
(b) contacting the methylated ester ketoside of Neu5Ac provided in step (a) with acetone and p-toluenesulfonic acid, to form an 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac;
(c) blocking the 4-hydroxyl group of the 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac with a blocking group to form a blocked 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac;
(d) contacting the blocked 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac provided in step (c) with methyl iodide and a mixture of barium oxide and barium hydroxide to form a 7-O-methyl blocked 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac;
(e) deblocking the 7-O-methyl blocked 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac to form a 7-O-methyl 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac;
(f) selectively removing the 8,9-isopropylidene and C2 methyl groups from the ketoside product obtained in step (e) and acetylating the anomeric, 7-, 8- and 9-hydroxyl groups of the resulting intermediate;
(g) activating the anomeric carbon of the product obtained in step (f) by converting the acetate to chloride;
(h) contacting the activated chloride product provided in step (g) with a suspension of a derivative of 5-bromo-3-indolol and potassium tert-butoxide in a solvent to form a chromogenic ketoside of a 7-O-methyl methyl ester of Neu5Ac, acetylated at the 7-, 8- and 9-hydroxyls; and
(i) deblocking the acetyl groups at the 7-, 8- and 9-hydroxyl groups and the methyl ester from the chromogenic ketoside of Neu5Ac obtained in step (h), to form the 7-O-methyl chromogenic ketoside of Neu5Ac.
Additionally the invention discloses a method of preparing a 7-O-methyl chromogenic ketoside of N-acetylneuraminic acid (Neu5Ac) that includes, in order, the following steps:
(a) contacting Neu5Ac with methanol and the protonated form of a cation exchange resin to methylate the C1 carboxyl group and the C2 hydroxyl group, forming a methyl ester methyl ketoside of Neu5Ac;
(b) contacting the methylated ester ketoside of Neu5Ac provided in step (a) with
(i) an acid chloride of an organic sulfonic acid, and
(ii) treating the result obtained in step (b) (i) with a composition containing sodium methoxide and methanol, providing an 8,9-epoxide methyl ester methyl ketoside of Neu5Ac;
(c) blocking the 4-hydroxyl group of the 8,9-epoxide methyl ester methyl ketoside of Neu5Ac with a blocking group to form a blocked 8,9-epoxide methyl ester methyl ketoside of Neu5Ac;
(d) methylating the 7-hydroxyl group of the blocked 8,9-epoxide methylated ester ketoside of Neu5Ac provided in step (c) by a process chosen from the group consisting of
(i) contacting the epoxide ketoside with a composition containing methyl iodide and silver oxide,
(ii) contacting the epoxide ketoside with a composition containing dimethylsulfate and sodium hydride, and
(iii) a sequential combination of step (c)(i) and step (c)(ii), providing a 7-O-methyl blocked 8,9-epoxide methylated ester ketoside of Neu5Ac;
(e) deblocking the 7-O-blocked methyl blocked 8,9-epoxide methyl ester methyl ketoside of Neu5Ac to form a 7-O-methyl 8,9-isopropylidene methyl ester methyl ketoside of Neu5Ac;
(f) selectively removing the 8,9-epoxide and C2 methyl groups of the ketoside product obtained in step (e) and acetylating the anomeric, 4-, 8- and 9-hydroxyl groups of the resulting intermediate;
(g) activating the anomeric carbon of the product obtained in step (f) by converting the acetate to chloride;
(h) contacting the activated chloride product provided in step (g) with a suspension of a derivative of 5-bromo-3-indolol and potassium tert-butoxide in a solvent to form a chromogenic ketoside of a 7-O-methyl methyl ester of Neu5Ac, acetylated at the 4-, 8- and 9-hydroxyls; and
(i) deblocking the acetyl groups at the 4-, 8- and 9-hydroxyl groups and the methyl ester from the chromogenic ketoside of Neu5Ac obtained in step (h), to form the 7-O-methyl chromogenic ketoside of Neu5Ac.